DNA sequencing can now be performed automatically by machine. See generally, W. Ansorge et al., 13 J. Biochem. Biophys. Methods 315-323 (1986). The disclosures of this publication and the disclosures of all other publications recited herein are incorporated by reference as if fully set forth herein.
One type of commercially available machine for automated sequencing is the A.L.F. DNA Sequencer, sold by Pharmacia LKB Biotechnology. This sequencer uses a primer, which is a small oligonucleotide of known sequence. The primer hybridizes to a section of a DNA molecule that is to be sequenced and thereby creates a short double-stranded region that enables DNA polymerase to replicate the single-stranded portion of the DNA. In accordance with the Sanger dideoxy sequencing method, four types of dideoxy terminators are separately added during synthesis to create different length fragments, and these fragments are then separated on a gel by length. The gel positions of the fragments provide sequence information.
This method of automated sequencing relies on the addition of labels to the primer so that after separation on the gel the position of the labels can automatically be read by the machine. Labelled primers include those with fluorescent, bioreactive, chemiluminescent, or photolabile labels.
Labels have usually been attached to an oligonucleotide in a multi-step procedure. The first step requires synthesis of an oligonucleotide bearing a protected amine by attaching a protected phosphoramidite/linker to the oligonucleotide. Then, the resulting compound is deprotected. After that, the compound is linked to a fluorescent label.
This procedure requires partial purification of the deprotected oligonucleotide, reaction with the label, removal of the excess reagent, and then purification of the labelled oligonucleotide. The overall process is tedious, and usually requires two days to produce a labelled oligonucleotide. Additionally, there are yield losses at each step in the multi-step process. Further, this work must normally be done by the user of the automated sequencer since the user usually wants to custom design an appropriate short nucleotide to prime a sequence of interest.
To avoid these problems, the art prepared label-linked phosphoramidites which could later be reacted with oligonucleotides. Amidites linked to a chemically inert bathophenanthroline-ruthenium complex, W. Bannwarth, et al., 30 Tet. Let. 1513-1516 (1989) have been prepared. Biotin-containing amidites have also been prepared. A. Alves, et al., 30 Tet. Let. 3089-3092 (1989). Also, amidites linked to dinitrophenyl, dansyl, and pyrenyl labels have been prepared. A. Roget et al., 17 Nuc. Acids Res. 7643-7651 (1989). European Patent Application 89116946.8 (Publication No. 359,255) discloses a xanthene dye-linked amidite.
However, fluoresceins are highly preferred labels. They have exceptionally high fluorescent emission/mol, excellent solubility, relatively low cost, and other useful characteristics. F. Schubert et al., 18 Nucl. Acids Res. 3427 (1990) attempted the synthesis of a phosphoramidite linked to fluorescein, albeit through one of the fluorescein phenolic hydroxyls. Unfortunately, this approach significantly reduced fluorescence and shifted absorption and emission wavelengths from desired regions.
Thus, the normally used method to obtain fluorescein labelled oligonucleotides still is the multistep approach, an example of which is described in FIG. 1. Addition of linker phosphoramidite A to the 5' end of an oligonucleotide on a DNA synthesizer yields B. After deprotection and desalting of B to yield D, the active nucleophile in D reacts with fluorescein isothiocyanate or carboxyfluorescein, N-hydroxysuccinimide (NHS) ester to yield a labelled oligonucleotide in which fluorescein is bound to the oligonucleotide through a thiourea or amide group and a six carbon linker. This procedure suffers from the problems described above for multi-step approaches, but yields a product with the characteristic fluorescein absorption and emission wavelengths.
Complicating matters is the fact that fluorescein has two active oxygen sites that when unprotected will interfere with a phosphoramidite attempting to link to fluorescein. Moreover, linkages to phosphoramidites can be adversely affected by deprotection conditions used for many protecting groups. Also, many conditions will affect or cause a reaction with a carboxyl group on the fluorescein ring.
As such, a need exists for labelled fluorescein phosphoramidites having good stability characteristics, which can readily be synthesized at high yields, and which confer high fluorescent emission/mol at desired wavelengths when linked to oligonucleotides.